Actin cytoskeleton regulates stretch-activated Ca²⁺ influx in human pulmonary microvascular endothelial cells

During high tidal volume mechanical ventilation in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), regions of the lung are exposed to excessive stretch, causing inflammatory responses and further lung damage. In this study, the effects of mechanical stretch on intracellular Ca²⁺ concentration ([Ca²⁺]_i), which regulates a variety of endothelial properties, were investigated in human pulmonary microvascular endothelial cells (HPMVECs). HPMVECs grown on fibronectin-coated silicon chambers were exposed to uniaxial stretching, using a cell-stretching apparatus. After stretching and subsequent unloading, [Ca²⁺] _i, as measuredbyfura-2fluorescence,wastransiently increased inastrain amplitude-dependent manner. The elevation of [Ca²⁺]_i induced by stretch was not evident in the Ca²⁺-free solution and was blocked by Gd31, a stretch-activated channel inhibitor, or ruthenium red, a transient receptor potential vanilloid inhibitor. The disruption of actin polymerization with cytochalasin D inhibited the stretch-induced elevation of [Ca²⁺]_i. In contrast, increases in [Ca²⁺] _i induced by thapsigargin or thrombin were not affected by cytochalasin D. Increased actin polymerization with sphingosine-1-phosphate or jasplakinolide enhanced the stretch-induced elevation of [Ca²⁺] _i. A simple network model of the cytoskeleton was also developed in support of the notion that actin stress fibers are required for efficient force transmission to open stretch-activated Ca²⁺ channels. In conclusion, mechanical stretch activates Ca²⁺ influx via stretch-activated channels which are tightly regulated by the actin cytoskeleton differentfrom other Ca²⁺ influx pathways such as receptor-operated and store-operated Ca²⁺ entries in HPMVECs. These results suggest that abnormal Ca²⁺ homeostasis because of excessive mechanical stretch during mechanical ventilation may play a role in the progression of ALI/ARDS.